RNA viruses represent the largest class of existing and emerging human pathogens, causing life-threatening gastrointestinal, respiratory, hemorrhagic, and neurological diseases. Although RNA viruses can vary widely in their genome sequences, virion architectures, tissue tropism, and pathological manifestations, they all encode a specialized enzyme called an RNA-dependent RNA polymerase (RdRp). The RdRp is critical for viral replication, as it mediates all stages of viral RNA synthesis. Yet, the activity of the vral RdRp must be regulated during infection so that RNA synthesis can be coordinated with other steps in the viral lifecycle. In many cases, such regulation is mediated by the binding of an effector protein to an allosteric site on the RdRp surface, which is physically distinct from the active site. Binding of the allosteric site by the effector protein induces a series of intra-molecular conformational changes in the RdRp that culminate at the active site to modulate enzyme function. The overall objective of this proposal is to gain mechanistic insight into allosteric RdRp regulation using rotavirus as a structurally- and functionally-tractable experimental system. Rotavirus is a double-stranded RNA virus that causes severe gastroenteritis in young children. The activity of the rotavirus RdRp (VP1) requires that it be directly engaged by the core shell effector protein (VP2). However, key gaps in knowledge exist about (i) which surface-exposed VP1 residues comprise the allosteric activation site and (ii) which buried VP1 residues transmit the allosteric signal from the surface to the active site. Two integrated, yet independent, specific aims are proposed to help close these gaps in knowledge. In Aim 1, a structure-guided, gain-of-function biochemical approach will be used to map the precise residues that comprise the VP1 allosteric activation site. Specifically, chimeric and point mutant VP1 proteins will be engineered and tested for their capacity to mediate in vitro RNA synthesis in the presence of cognate and non-cognate VP2. In Aim 2, in silico amino acid co-variation analysis and molecular dynamic simulations will be employed to identify buried VP1 residues that may transmit the allosteric signal. These residues will then be validated using mutant VP1 proteins and in vitro RNA synthesis assays. Upon completion of this work, it is expected that an auto-activated VP1 mutant will have been created, and the precise amino acid residues involved in VP1 allosteric activation will have been defined. This proposal is innovative because it uses sequence-based and structure-function methodologies to investigate original ideas about how the enzymatic activity of VP1 is regulated by VP2. The work is significant because it will reveal features of the rotavirus RdRp that are shared with those of other pathogenic RNA viruses, which may in-turn foster the development of allosteric antiviral drugs to treat and prevent viral diseases.